1. Volume 96, Issue 6, Pages 1358-1372.e4 (December 2017)
Cholinergic Projections to the Substantia Nigra Pars Reticulata Inhibit Dopamine Modulation of Basal Ganglia through the M4 Muscarinic Receptor 
Mark S. Moehle, Tristano Pancani, Nellie Byun, Samantha E. Yohn, George H. Wilson, Johnathan W. Dickerson, Daniel H. Remke, Zixiu Xiang, Colleen M. Niswender, Jürgen Wess, Carrie K. Jones, Craig W. Lindsley, Jerri M. Rook, P. Jeffrey Conn 
Neuron 
Volume 96, Issue 6, Pages e4 (December 2017)
DOI: /j.neuron
Copyright © 2017 Elsevier Inc. Terms and Conditions

2. Figure 1
D1 Agonists Increase Locomotion and Are Reversed by Administration of the M4 PAM VU
(A) WT animals were injected with M4 PAM VU (30 mg/kg, i.p., 10% Tween 80) 90 min after being placed in locomotion chambers. Thirty minutes later, D1 agonist SKF82958 was administered (1 mg/kg, i.p., sterile water). Activity was then recorded for an additional 60 min and reported as distance in centimeters (cm) per 5 min bins.
(B) Dose-response relationship of 3, 10, and 30 mg/kg VU (i.p., 10% Tween 80) in WT mice following the injection pattern in (A). Data are the total distance moved in cm after the injection of amphetamine.
(C) D1-M4 KO mice were injected with M4 PAM VU , SKF82958, and/or vehicle as in (A).
(D) Dose-response relationship of 3, 10, and 30 mg/kg VU (i.p., 10% Tween 80) in D1-M4 KO mice following the same pattern in (A). VU is unable to block D1-induced hyperlocomotion in D1-M4 KO mice.
See also Figure S1. Data are mean ± SEM with n = 8–12 per treatment group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not statistically significant by one-way ANOVA followed by Tukey’s post hoc test.
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3. Figure 2
D1 Agonists Increase GABA Release in the SNr, and This Effect Is Reversed by Muscarinic Activation
(A) Representative mIPSC traces from GABAergic cells of the SNr treated with 10 μM SKF82958 and 10 μM Oxo-M, 10 μM VU , or 2 μM Oxo-M and 5 μM VU in WT mice.
(B) Cumulative probability plots of traces in (A).
(C) Graph of data represented in (A) and (B). Positive modulation corresponds to an increased mIPSC frequency and negative modulation corresponds to decreased mIPSC frequency as compared to baseline.
(D) Representative mIPSC traces from mice with ChR2 expressed under the choline acetyltransferase (ChAT-ChR2) promoter that were then optogenetically stimulated with 10 Hz, 5 ms pulses of 470 nm light to release ACh followed by treatment with 5 μM SKF82958, 5 μM M4 PAM VU , or a combination of these.
(E) Cumulative probability plots of traces in (D).
(F) Summary of data represented in (D) and (E). Positive modulation corresponds to an increased mIPSC frequency and negative modulations correspond to decreased mIPSC frequency as compared to baseline.
See also Figures S2–S4. Data are mean ± SEM with n = 8–11 per group in (A)–(C) and n = 8–14 per group in (D)–(F). ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not statistically significant by one-way ANOVA followed by Tukey’s post hoc test.
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4. Figure 3
M4 Activation on D1-SPN Terminals in the SNr Blocks D1-Evoked GABA Release in the SNr
(A) Representative mIPSC traces from GABAergic cells of the SNr in slices from D1-M4 KO mice treated with 10 μM SKF82958 and 10 μM Oxo-M, 10 μM VU , or 2 μM Oxo-M and 5 μM VU , or from D1-M4 KO mice injected with AAV2-hM4Di DREADD then treated with 10 μM SKF82958 and 10 μM CNO or CNO alone.
(B) Cumulative probability plots of traces in (A).
(C) Summary of data represented in (A) and (B). Positive modulation corresponds to an increased mIPSC frequency and negative modulation corresponds to decreased mIPSC frequency as compared to baseline.
See also Figure S4. Data are mean ± SEM with n = 8–10 per group. NS, not statistically significant by one-way ANOVA followed by Tukey’s post hoc test.
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5. Figure 4 M4 Receptor Activity May Tonically Inhibit D1-SPNs
(A) Representative mIPSC traces recorded from GABAergic cells of the SNr from WT mice, mice that express Cre-recombinase under the D1 promoter (D1-Cre), or D1-M4 KO mice.
(B) Graph of baseline mIPSC frequency from (A).
(C) Representative mIPSC traces from WT or D1-M4 KO from GABAergic cells of the SNr treated with 50 μM scopolamine, 1 μM of the highly selective M4 antagonist mamba toxin-3 (MT-3), or 10 μM VU (VU154).
(D) Cumulative probability plots of traces in (A).
(E) Summary of data represented in (C) and (D).
(F) Representative images from ChAT-Cre animals injected with an AAV control virus or constitutively active caspase3 construct. Sections from the hindbrain were made and stained with an anti-vesicular choline acetyltransferase antibody and appropriate secondary antibody (green). Widefield images were taken using a 5× objective, then stitched together. Images (20×) were taken on the border of the LDT and PPN to visualize both structures. Caspase3-injected animals showed fewer VAChT-positive neurons than control virus-injected animals.
(G) Summary of ex vivo electrophysiological changes in mIPSC frequency from control or caspase3-injected ChAT-Cre animals after bath application of 1 μM MT-3.
(H) Graph of baseline mIPSC frequency from GABAergic cells of the SNr from control or caspase3-injected ChAT-Cre animals. Positive modulation corresponds to an increased mIPSC frequency and negative modulation corresponds to decreased mIPSC frequency as compared to baseline.
Data are mean ± SEM with n = 15–20 per group (A and B), n = 8–12 (C and D), and n = 6–8 (C and D). ∗∗∗p < by one-way ANOVA followed by Tukey’s post hoc test (A and B), ∗∗∗p < 0.001, ∗p < 0.05 by Student’s t test (C–E), ∗p < 0.05 by Mann-Whitney test (F–H).
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6. Figure 5
Adenylyl Cyclase Activation in D1-SPN Terminals Mimics D1 Agonists and Is Blocked by M4 Activation
(A) Diagram of AAV2-β2 Opto XR viral injection into the dorsolateral striatum and coronal sectioning technique of the SNr.
(B) Confocal microscopy images of mice with the fluorescent protein td-Tomato expressed under the D1 promoter (D1-td-Tomato, red) injected with AAV2-β2 Opto XR viral construct, which has an enhanced yellow fluorescent protein (eYFP) reporter (green). While the expression of the viral construct is in both D1 and non-D1 structures in the striatum, due to sectioning technique, the viral construct (green) has near-complete co-localization (yellow) with the D1 reporter (red) in the SNr.
(C) Representative mIPSC traces of GABAergic cells of the SNr from WT animals expressing the β2 Opto XR or eYFP control construct before and after optical stimulation and treatment with 10 μM Oxo-M or 2 μM Oxo-M and 5 μM VU
(D) Summary of data represented in (C). Positive modulation corresponds to an increased mIPSC frequency and negative modulation corresponds to decreased mIPSC frequency as compared to baseline.
(E) Representative mIPSC traces of GABAergic cells of the SNr from D1-M4 KO mice expressing the β2 Opto XR or eYFP control construct before and after optical stimulation and treated with 10 μM Oxo-M or 2 μM Oxo-M and 5 μM VU
(F) Summary of data represented in (E). Positive modulation corresponds to an increased mIPSC frequency and negative modulation corresponds to decreased mIPSC frequency as compared to baseline.
See also Figure S5. Data are mean ± SEM with n = 8–10 per group (C and D) and n = 8–14 per group (E and F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, not statistically significant by one-way ANOVA followed by Tukey’s post hoc test.
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7. Figure 6
M4 Activity Suppresses D1 Activation at the Level of the SNr and May Inhibit D1 Signaling in a Synapse-Specific Manner
(A) Structural T2-weighted MRI template images and CBV activation maps across nine slices (caudal to rostral) of male Sprague-Dawley rats anesthetized and then treated with vehicle (i.p., 10% Tween 80) or 30 mg/kg VU (i.p., 10% Tween 80) and then 1 mg/kg SKF82958 (i.p., sterile water). In the group activation maps, the red to yellow to red bar range represents increased CBV, indicating increased neuronal activity, while blue to purple color bar range represents decreased CBV, indicating decreased neuronal activity.
(B–G) Time courses and bar graphs of CBV changes after SKF82958 injection in rats pretreated with vehicle or M4 PAM VU from the (B) SNr, (C) striatum, (D) motor cortex, (E) sensory cortex, (F) hippocampus, or (G) cingulate cortex. Time course graphs show fractional changes in CBV (ΔCBV(t) over baseline [CBVo]) before SKF82958 injection, which was injected at minute 10. For the bar graphs, fractional CBV values were averaged between 20 and 30 min for each animal.
See also Figures S6 and S7. Data are mean ± SEM with n = 6–8 per group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; NS, non-significant by Wilcoxon matched pairs and rank test.
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8. Figure 7
Muscarinic Activation in the SNr Is Sufficient to Block Locomotion
(A) WT mice were cannulated with the cannula sitting just above the SNr. Mice were pre-treated with the M1 antagonist VU (3 mg/kg, i.p., 10% Tween 80) to prevent the influence of M1 activity on rotational behaviors 15 min prior to microinjection (MI). Mice were then injected in the SNr through the implanted cannula with 0.5 mg/mL Oxo-M in l μL sterile water or 1 μL sterile water alone. Animals that were injected with 0.3 mg/kg SKF89258 (i.p., sterile water) were injected 15 min prior to microinjection. Rotations were observed and scored by an experimenter blinded to conditions for 15 min after microinjection. Data represent total ipsilateral rotations.
(B) D1-M4 KO or littermate control mice that were cannulated in the SNr and microinjected with Oxo-M as in (A) and their rotations observed.
(C) WT or D1-M4 KO mice were bi-laterally cannulated in the SNr and allowed to recover for 1 week. Mice were placed in an open field chamber and allowed to habituate for 90 min. After habituation, mice were bi-laterally microinjected with 1 μL of 3 mg/mL scopolamine (sterile water) or sterile water alone.
Data are shown as total distance traveled (cm) after microinjection. Data are mean ± SEM with n = 8 per group (A), n = 10–12 per group (B), and n = 10–12 per group (C). ∗p < 0.05, ∗∗p < 0.01 by Kruskal-Wallis test with Dunnett’s compare all columns post-test.
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9. Figure 8 Model of D1 and M4 Regulation of the Direct Pathway
(A) M4 activation in the striatum has complex actions on the circuitry of the BG. M4 activation specifically on D1-SPNs causes the release of an endocannabinoid (purple circles), which acts on cannabinoid receptor 2 (CB2) receptors on DA terminals from the SNc to cause a sustained inhibition of DA release (X markers in DA terminals). In glutamatergic projections from the cortex and thalamus, M4 activation has been shown to decrease excitatory transmission and promote long-term depression in the striatum. M4 activation on cholinergic interneurons is suggested to decrease tonic firing and ACh release.
(B) In the SNr, M4 decreases GABA release probability through inhibiting AC and downstream cAMP signaling. Together, M4 activation on D1-SPNs in both the striatum and SNr is predicted to provide an efficient brake on D1-SPN activity.
Neuron  , e4DOI: ( /j.neuron )
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